Expression of human proteins at the Southeast Collaboratory for Structural Genomics

J Struct Funct Genomics. 2004;5(1-2):159-65. doi: 10.1023/B:JSFG.0000029202.77832.34.

Abstract

The human protein production group at the Southeast Collaboratory for Structural Genomics is charged with producing human proteins for both X-ray crystallography and NMR structural studies. Eukaryotic, and human proteins in particular, are notoriously difficult to express in bacterial systems. For various reasons, T7-based expression often results in protein expressed in an insoluble form. Overcoming this requires either introduction of a step to screen expression conditions or inclusion of a troublesome refolding step during purification. Our laboratory uses a trc-based expression vector that addresses many of the difficulties of the commonly used T7-based expression systems. Proteins expressed under the trc promoter, a weak promoter compared to the strong T7 promoter, are produced in a soluble form and include necessary cofactors. The details of this system will be discussed.

Publication types

  • Comparative Study

MeSH terms

  • Cloning, Molecular
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Expression
  • Genetic Vectors
  • Genomics / methods*
  • Georgia
  • Humans
  • Laboratories
  • Molecular Structure
  • Promoter Regions, Genetic
  • Proteomics / methods*
  • Recombinant Proteins / biosynthesis*
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / isolation & purification
  • Solubility

Substances

  • Recombinant Proteins